Project Summary/Abstract Soils and waters with high levels of toxic metal(loid)s such as cadmium, lead, arsenic and mercury are detrimental to human and environmental health. These 4 heavy metal(loid)s are among the Superfund's top 7 priority hazardous substances. Many human diseases have been attributed to environmental contamination by heavy metals, including cancers and neurological disorders. Research and applications indicate that uptake of heavy metals into plant roots and accumulation of heavy metals could provide a cost effective approach for toxic metal removal and bioremediation of heavy metal-laden soils and waters. In recent research we have made major advances at understanding key mechanisms that function in heavy metal detoxification, transport and accumulation in plants. However important genes and pathways that function in heavy metal over- accumulation in plants remain to be identified. We will combine powerful genomic, genetic, biochemical and engineering approaches to test new central hypotheses by pursuing the following Specific Aims: Aim I. The regulatory mechanisms, transcription factors (TFs), and transcriptional network that mediate rapid heavy metal(loid)-induced transcriptional responses in plants remain largely unknown. Using a luciferase- based cadmium- and arsenic-induced reporter mutant screening approach we have isolated mutants in rapid Cd- and As-induced gene expression. New mutants in major Cd-/As-dependent repression and induction loci will be characterized and the underlying genes isolated and their functions determined. Collaborative research with Geoffrey Chang (Project 5) will pursue development of cost-effective innovative heavy metal toxicant nano-reporters in plants. Aim II. The many genetic redundancies in plant genomes cause major limitations in heavy metal response gene discovery. To address redundant gene function on a systems biology scale we have designed a genomic scale artificial microRNA (amiRNA) library for genome-wide knockdown of homologous gene family members which is leading to discovery of new genes and will be used to characterize key plant genes and network mechanisms that function in heavy metal accumulation, resistance and remediation. Aim III: Using genes identified in Specific Aims I and II and previous research, gene-stacking will be used to generate plants and investigate their enhanced heavy metal accumulation and root sequestration (phytostabilization) potential. Furthermore, by genomic investigation of plants that are being used for phytostabilization at semi-arid Superfund sites, the above advances will be used in collaboration with the University of Arizona Superfund Research Center to uncover mechanisms that render plants suitable for phytostabilization of toxic metal(loid)s. The proposed research will be leveraged to develop technologies for avoiding the growing problem of accumulation of heavy metals and arsenic in edible plant tissues.